Throughout this application, various publications may be referenced by Arabic numerals in brackets. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
This invention discloses a sterol-responsive region of the human ABC1 promoter, and shows that it is activated by hydroxysterols and 9-cis-retinoic acid, implicating a mechanism of activation involving LXR/RXR heterodimers. Also disclosed is the functionally active form of the ABC1 cDNA.
Plasma HDL-C levels are inversely related to the incidence of coronary artery disease (1). Two genetic diseases illustrate this phenomenon: the rare Tangier Disease and the more common familial HDL deficiency. Tangier disease is characterized by an extremely low concentration of circulating HDL and the accumulation of cholesteryl esters in tonsils, liver, spleen and intestinal mucosa, mostly in macrophage foam cells (2). Patients with familial HDL deficiency exhibit a low concentration of HDL particles and an increased risk of coronary artery disease (3). A common explanation for the cardioprotective effect of HDL is the major role they play in reverse cholesterol transport (4). It is commonly accepted that the efflux of cholesterol from cells is due to two different pathways: the first is passive and promotes efflux from the cell membrane to HDL, and the second is energy-dependent and apolipoprotein-mediated (5). The latter was characterized in fibroblasts and macrophages, and involves lipid-poor or lipid-free apolipoproteins such as apoA-I, apoA-II and apo-E (5). This active pathway has been reported to be defective in both Tangier disease and familial HDL deficiency (6-7). It was recently shown that ABC1 is a key gene in this process (8) and that mutations in this gene are the major cause of Tangier disease and familial HDL deficiency (3, 9-14).
ABC1 (ABCA1) belongs to the large ATP-Binding Cassette transporter family. These transmembrane proteins transport many substrates across membranes thanks to a channel-like topology (15-16). The human ABC1 gene was assigned to chromosome 9q31, spanning a minimum of 70 kb and containing at least 49 exons (11, 13, 17). While its expression is ubiquitous, the highest levels of human or murine mRNAs were found in placenta, liver, lung, adrenal glands and fetal tissues (18, 19). The predicted human protein contains 2201 amino acids (220 kDa) (18).
The expression of hABC1 is induced during differentiation of human monocytes to macrophages and as a result of cholesterol loading. In human macrophages, both the protein and the mRNA are upregulated in the presence of acetylated LDL (acLDL), and downregulated by cholesterol unloading via HDL3 (18). While the cholesterol-mediated downregulation of genes involved in cholesterol uptake or biosynthesis is well understood (20), mechanisms of sterol-mediated upregulation of gene expression are more poorly understood. Two families of nuclear receptors are known to be activated by oxysterols and to mediate a positive response by binding to specific DNA elements: Liver-X-Receptor (LXR) and Steroidogenic Factor 1 (SF1) (21), (22), (23), (24). SF1 acts as a monomer and has been implicated, for example, in the regulation of steroidogenic acute regulatory protein gene expression (StAR) (23). Recently, two different genes involved in reverse cholesterol transport pathways, cyp7a (21) and CETP (22), have been shown to be upregulated by the heterodimer LXR-RXR (Liver-X-Receptor and Retinoid-X-Receptor). This suggests the hypothesis that LXRs might coordinate different steps of these pathways (22). LXRxcex1 (NR1H3) and LXRxcex2 (NR1H2) heterodimerize with their partner RXR to upregulate genes through binding sites composed of direct repeats (DR) spaced by 4 (LXRxcex1 and LXRxcex2) or 1 nucleotide (LXRxcex2) (25-27). The dimer can be activated by both the ligands of RXR (retinoids) and LXR (oxysterols), separately or together (26, 28, 29).
However, none of the cited references disclose the sequence of the hABC1 promoter, nor do they define its function or activation by transcription factors or small organic molecules.
This disclosure provides an isolated human ABC1 promoter capable of directing transcription of a heterologous coding sequence positioned downstream therefrom, wherein the promoter is:
(a) a promoter comprising the nucleotide sequence shown in SEQ ID NO: 1;
(b) a promoter comprising a nucleotide sequence functionally equivalent to the nucleotide sequence shown in SEQ ID NO: 1; or
(c) a promoter comprising a nucleotide sequence that hybridizes to a sequence complementary to the promoter of (a) or (b) in a Southern hybridization reaction performed under stringent conditions.
This disclosure also provides a recombinant expression construct effective in directing the transcription of a selected coding sequence which comprises:
(a) a human ABC1 promoter nucleotide sequence according to claim 1; and
(b) a coding sequence operably linked to the promoter, whereby the coding sequence can be transcribed and translated in a host cell, and the promoter is heterologous to the coding sequence.
This disclosure also provides a method for expressing foreign DNA in a host cell which comprises introducing into the host cell a gene transfer vector comprising the discussed ABC1 promoter operably linked to a foreign DNA encoding a desired polypeptide or RNA, wherein said foreign DNA is expressed.
The disclosure also provides a method of determining whether a chemical not previously known to be a modulator of the human ABC1 gene transcriptionally modulates the expression of the human ABC1 gene which comprises:
(a) contacting a sample which contains a predefined number of identical eucaryotic cells with a predetermined concentration of the chemical to be tested, each cell comprising a DNA construct consisting essentially of in 5xe2x80x2 to 3xe2x80x2 order,
(i) a modulatable transcriptional regulatory sequence of the ABC1 gene,
(ii) the ABC1 promoter of claim 1, and
(iii) a reporter gene which expresses a polypeptide that produces a detectable signal, coupled to, and under the control of, the ABC1 promoter, under conditions wherein the chemical if capable of acting as a transcriptional modulator of the ABC1 gene, causes a detectable signal to be produced by the polypeptide expressed by the reporter gene;
(b) quantitatively determining the amount of the signal produced in (a); and
(c) comparing the amount of signal determined in (b) with the amount of signal produced and detected in the absence of any chemical being tested or with the amount of signal produced and detected upon contacting the sample in (a) with other chemicals, thereby identifing the test chemical as a chemical which causes a change in the amount of detectable signal produced by the polypeptide expressed by the reporter gene, and determining whether the test chemical specifically transcriptionally modulates expression of the human ABC1 gene.
This disclosure also provides a method of treating atherosclerosis in a subject which comprises administering to the subject a therapeutically effective amount of a chemical selected by the method discussed above to modulate expression of the human ABC1 gene.
This disclosure also provides a transgenic non-human mammal whose germ or somatic cells contain the promoter described herein introduced into the mammal, or an ancestor thereof, at an embryonic stage. The mammal may be a mouse.
This disclosure also provides a compound which modulates expression of the human ABC1 gene, which has been identified by the method discussed above.
This disclosure also provides an isolated human ABC1 gene comprising six exons and the promoter described.
Thus, disclosed is the sequence of the hABC1 promoter and its 5xe2x80x2 untranslated region, with 3 new exons, differentially expressed and carrying two alternative start codons. This promoter is active in macrophages. Also, the full length cDNA, incorporating the 3 new exons plus the published cDNA sequence, is active in promoting efflux of cholesterol from cells, while the published cDNA sequence alone is not. Also disclosed is that RXRxcex1 and LXRxcex1 or LXRxcex2 are able to activate in vitro this promoter and the response is increased by oxysterols and/or 9-cis-retinoic acid (9CRA).